Sequence Mapping of mRNA Digests Using the Xevo™ MRT Mass Spectrometer and waters_connect™ MAP Sequence 2.0 Application

Significance of the topic

Accurate characterization of mRNA sequence and chemical modifications is essential for the development, quality control, and regulatory approval of mRNA-based therapeutics and vaccines. Conventional sequencing methods such as Sanger and next-generation sequencing (NGS) often fail to detect sequence modifications or degradation products. Liquid chromatography–mass spectrometry (LC-MS) workflows, when coupled with specialized digestion enzymes and automated informatics, can overcome these limitations by delivering high specificity, sensitivity, and coverage for oligonucleotide mapping.

Study objectives and overview

This application note outlines an integrated workflow for sequence mapping of Firefly luciferase mRNA (Fluc) using complementary ribonucleases and UPLC-MSE data acquisition on the Xevo™ MRT Q-Tof mass spectrometer. The goals are to demonstrate higher overall sequence coverage through the use of two novel RNase T2 enzymes (RapiZyme™ MC1 and Cusativin) alongside conventional RNase T1, and to streamline data interpretation using the waters_connect™ MAP Sequence App v2.0.

Methodology

• Sample preparation: Fluc mRNA (1,813 nt plus Cap1 and poly(A) tail) was denatured at 90 °C and digested separately with RNase T1, RapiZyme MC1 (pH 8.0, 30 °C), and RapiZyme Cusativin (pH 9.0, 30 °C), with controlled enzyme amounts to promote missed cleavages.
• LC-MSE acquisition: 5 µL injections on an ACQUITY Premier Oligonucleotide BEH C18 column at 70 °C, with a binary gradient of DIPEA/HFIP aqueous and acetonitrile buffers; data collected in negative ESI mode at 2 Hz, low-energy and high-energy ramps for MS^E.
• Informatics: waters_connect™ platform (v4.1.0.17) using the SYNTHETIC Library App v2.0 to input the Fluc sequence and predict in silico digest products; MAP Sequence App v2.0 for automated precursor and fragment matching, sequence coverage calculation, and report generation.

Instrumental details

• UPLC: Waters ACQUITY Premier system with an Oligonucleotide BEH C18, 1.7 µm, 2.1 × 150 mm column, 400 µL/min flow.
• Mass spectrometer: Xevo™ MRT multi-reflecting time-of-flight Q-Tof, ESI(–), capillary 1.5 kV, cone 40 V, source 120 °C, desolvation 550 °C, gas flows: cone gas 0 L/h, desolvation 1000 L/h; m/z range 50–4000.

Results and Discussion

• Enzyme specificity and coverage: RNase T1 (G-specific) produced many short, often isobaric fragments, yielding ~33 % unique coverage; RapiZyme MC1 (cleaves at A_U, C_U, U_U motifs) gave ~82 % coverage; Cusativin (C-specific) gave ~73 % coverage.
• Multi-enzyme strategy: combining the three digest maps increased aggregated coverage to ~95 %, illustrating the benefit of overlapping digestion products.
• Informatics resolution: MAP Sequence v2.0 matched monoisotopic masses within 5 ppm and used high-energy fragment ions to differentiate isobaric and structural isomers, applying acceptance thresholds for isotope similarity and fragment-ion coverage.
• Case studies: Examples of 12-mer isomer pairs were automatically resolved based on fragment-ion coverage differences (100 % vs ~58 %), enhancing assignment confidence.

Benefits and practical applications

• Fully automated data processing minimizes manual intervention and subjectivity.
• High sequence coverage through complementary RNase T2 enzymes supports thorough mRNA characterization.
• Capability to detect and confirm sequence modifications and missed cleavage products.
• Adaptable for critical quality attribute (CQA) monitoring in mRNA therapeutics and vaccines.

Future trends and opportunities

Integration of this LC-MS mapping workflow with other mRNA quality assays (capping, poly(A) tail analysis), development of additional ribonucleases with novel specificities, expansion to chemically modified and longer mRNA constructs, and further advances in high-throughput, real-time informatics promise to accelerate biopharma development and regulatory compliance.

Conclusion

The combination of novel RNase T2 enzymes, high-resolution UPLC-MSE on the Xevo™ MRT platform, and the waters_connect™ MAP Sequence App v2.0 delivers a robust, high-coverage, and automated workflow for mRNA sequence mapping. This approach enhances confidence in primary structure confirmation and supports comprehensive CQA analysis for mRNA-based therapeutics.

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